Sulfhydryl groups of native myosin and of the myosin heavy chains from physarum polycephalum compared to vertebrate skeletal, smooth, and non-muscle myosins

Abstract

Although a previously reported analysis of Physarum myosin detected no cysteine residues in the molecule, the myosin ATPase activity was inhibited by p-chloromercuribenzoate. We have re-examined this apparently contradictory finding. We found highly purified plasmodial myosin to be very sensitive to N-ethylmaleimide inhibition of the K +, Ca 2+-activated ATPase. An estimate of the number of reactive sulfhydryls of the native myosin using Ellman's reagent showed only 1.5 mol 11 min-reactive sulfhydryl/mol as eompared to 4.5 for chicken breast myosin in 5min. 3H- and 14C-labelled N-ethylmaleimide was used to estimate the total sulfhydryls of the SDS-denatured heavy chains. Plasmodiai myosin heavy chains bound 10–13~ of the N-ethylmaleimide bound by chicken breast myosin heavy chalns. Smooth muscle myosin heavy chains as weli as heavy chains of embryonic chicken presumptive myoblats had 65–70% of the reactive groups of chicken myotube myosin heavy chains. Amino acid analyses of purified Physarum myosin showed that some preparations contained cysteic acid residues even before performic oxidation. After the performic oxidation a mean value of 3 mol cysteic acid per 10 5 g Physarum myosin was found, or less than half that reported for striated muscle myosin. Our results show that in the sulfhydryl-poor plasmodial myosin each heavy chain contains at least two sulfhydryls, and probably more, but that there is variable oxidation of the total sulfhydryls. It has been reported that plasmodial myosin lacks rapidly reacting sulfhydryl groups when tested with an ATP analogue which reacts with light chains of vertebrate muscle myosins. Therefore, the 1–2 sulfhydryls of plasmodial myosin which react rapidly with Ellman's reagent appear to be on the heavy chain. Our results also suggest that during development of myotubes changes occur in the myosin heavy chains.